EP0359873B1

EP0359873B1 - Method of treating mastitis and other staphylococcal infections

Info

Publication number: EP0359873B1
Application number: EP88308667A
Authority: EP
Inventors: Peter Blackburn; June Pollack
Original assignee: Applied Microbiology Inc
Current assignee: Applied Microbiology Inc
Priority date: 1988-08-29
Filing date: 1988-09-19
Publication date: 1993-09-15
Anticipated expiration: 2008-09-19
Also published as: DE3884200T2; EP0359873A1; AU623864B2; DE3884200D1; AU2179388A

Description

This invention relates to the treatment and prevention of staphylococcal infection and, more particularly but not exclusively, to the treatment and prevention of staphylococcal bovine mastitis.
Lysostaphin is a bacteriocin secreted by a single known strain of Staphylococcus simulans originally isolated and named Staphylococcus staphylolyticus by Schindler and Schuhardt. The production of lysostaphin by S. staphylolyticus has been described previously in U.S. Patent No. 3,278,378 issued October 11, 1966 and in Proceedings of the National Academy of Sciences, Vol. 51, pp. 414-421 (1964). The single organism S. staphylolyticus (NRRL B-2628) which produced lysostaphin was recently identified as a biovar of S. simulans by Sloan et al., Int. J. System. Bacteriol., Vol. 32, pp. 170-174 (1982). Since the name S. staphylolyticus is not on the Approved List of Bacterial Names, the organism producing lysostaphin has been redesignated as S. simulans.
Bacteriocins are proteins secreted by bacteria that kill and sometimes lyse related bacteria. For example, lysostaphin lyses and kills practically all known staphylococcal species but is inactive against bacteria of all other genera. Lysostaphin, isolated from culture filtrates of S. simulans (NRRL B-2628) grown according to published references, is an endopeptidase which cleaves the polyglycine cross-links of the peptidoglycan found in the cell walls of staphylococci. In addition, cultures that produce lysostaphin appear to be resistant to its activities while cultures grown under non-lysostaphin producing conditions are sensitive.
Previous studies have shown that lysostaphin can be produced by fermentation techniques wherein S. simulans is grown in liquid culture. Such fermentation techniques are described in U.S. Patent No. 3,278,378 issued October 11, 1966, and in Proceedings of the National Academy of Sciences, Vol. 51, pp. 414-421 (1964). Various improvements in the production of lysostaphin by fermentation techniques have also been made as documented in U.S. Patents Nos. 3,398,056, issued August 20, 1968, and 3,594,284, issued July 20, 1971. The latter two references disclose improvements to culture medium and inoculation techniques whereby the production of lysostaphin by fermentation can be accelerated and improved. Lysostaphin is produced by S. simulans during exponential growth as an inactive precursor. The proenzyme is converted to active mature enzyme by protease produced by stationary phase cultures of S. simulans.
In addition, lysostaphin can be produced by recombinant microorganisms, including strains of E. coli, Bacillus subtilis and B. sphaericus which express the lysostaphin gene. In contrast to the natural production, lysostaphin accumulates during exponential growth in the culture medium of recombinant lysostaphin producing strains as fully processed mature active enzyme and is free of staphylococcal immunogenic contaminants.
Bovine mastitis is a costly problem to the dairy industry, costing over $2 billion per year in the United States alone. The disease is estimated to affect 50 per cent of American dairy cows to some degree, leading to unusable milk, decreased milk production, and, in cases of severe infection, the death of the animal.
Mastitis is caused by infection of the milk glands, principally by Staphylococcus aureus or Streptococcus agalactiae, and to a lesser degree by E. coli and other gram-negative bacteria or combinations thereof. Most streptococcal infections have proven to be effectively treatable using conventional antibiotic therapy. Staphylococcal mastitis has, however, proven more difficult to cure.
Traditional prevention of bovine mastitis can involve a complex regimen of daily teat-dipping with a disinfectant solution, (See, J. S. McDonald, 6 Veterinary Clinics of North America Large Animal Practice 269 (1984)) and may, in some cases, involve antibiotic-containing teat dips. Routine antibiotic therapy must be approached with caution, however, to minimize selection for antibiotic resistant strains. When infection does occur, intramammary infusion of antibiotics is indicated. Antibiotic therapy of this kind can reduce the infection so that the milk produced is saleable, but it generally does not lead to complete elimination of the causative organism.
In the past, staphylococcal mastitis has shown a poor response to antibiotic therapy and a tendency for infections to recur and become chronic. Studies on mastitis have indicated that part of the problem in treating mastitis is that a significant number of staphylococci remain viable in the mammary gland within phagocytic polymorphonuclear neutrophil leukocytes (PMN). It is believed that the staphylococci within the PMN are protected from the effects of the antibiotic, and, when lysis of the leukocyte occurs, the phagocytized staphylococci may provide a renewed source of mastitis-producing staphylococcal regrowth.
Studies on the possible mechanism of antibiotic evasion of phagocytized staphylococci in mastitis treatment show that lysostaphin had been rejected as a candidate for destroying phagocytized staphylococci. Craven et al., 29 Research in Veterinary Science 57 (1980); Craven et al., 5 Comp. Immun. Microbial. Infect. Dis. 447 (1982)) Craven et al., 51 Journal of Dairy Research 513 (1984) Craven et al., 44 Am.J.Vet. Res. 709. In these experiments lysostaphin was used in vitro to destroy extracellular staphylococci prior to or while exposing the phagocytized staphylococci to various test antibiotics, including synthetic penicillins such as cloxacillin, and gentamicin or to lysostaphin. Craven et al.'s results strongly suggest that lysostaphin would have no effect on mastitis since intracellular staphylococci were still viable after 20 hours of incubation in a lysostaphin containing solution., 51 Journal of Dairy Research at 515-516, and Table 2.
Lysostaphin has also been reported to penetrate human monocytes. Since monocytes are a different cell type than PMNs, this human model is not likely to be applicable to the treatment of bovine mastitis (van den Broek et al., 21 Scand. J. Immunol 189 (1985)).
Lysostaphin has also been shown to be effective in the treatment of staphylococcal renal abscesses in mice, particularly when used in sequence with the administration of methicillin., Dixon et al., 41 Yale J. Biol. Med. 62 (1968).
In man lysostaphin has also been used as a therapeutic agent for treatment of chronic nasal staphylococcal infections (Quickel, Jr. et al., 22 Applied Microbiology 446 (1971)). In one case of a resistant staphylococcal infection, lysostaphin was given systemically (Stark et al., 291 Medical Intelligence 239 (1974)). In general, however, there has been great skepticism and reluctance in the medical and veterinary communities concerning the systemic administration of lysostaphin. Lysostaphin was considered to be too highly immunogenic to have general use for anything but topical applications.

SUMMARY OF THE INVENTION

It has now been found that lysostaphin can be formulated as a composition with surprising effectiveness for killing Staphylococci, especially to prevent and/or cure staphylococcal mastitis, even in its chronic form, without observed adverse immunogenic effects. The composition comprises lysostaphin, preferably in a concentration of at least 0.01mg/ml and least one cell wall-active antibiotic, especially penicillin, in a synergistic combination as recited in Claim 1. As a prophylactic, the composition can be introduced as part of a daily teat-dipping regimen. The composition preferably includes at least one other bacteriolytic agent such as mutanolysin, a bacteriocin produced by Streptococcus globisporus which is effective against streptococci; and lysozyme, a muralytic enzyme which hydrolyzes the polysaccharide backbone of the peptidoglycan in the cell walls of Gram positive and Gram negative bacteria. The formulation may also contain a chelating agent such as ethylenediamine tetraacetate (EDTA); and a mild surfactant, preferably in a concentration of from 0.1 to 1.0 percent, which has been found to potentiate the killing of the bacteria. Suitable mild surfactants include, inter alia, esters of polyoxyethylene sorbitan and fatty acids (Tween series), octylphenoxy polyethoxy ethanol (Triton-X series), n-octyl-β-D-glucopytranoside, n-octyl-β-D-thioglucopyranoside, n-decyl-β-D-glucopyranoside, n-dodecyl-β-D-glucopyranoside, and biologically occurring surfactants, e.g. fatty acids, glycerides, monoglycerides, deoxycholate and esters of deoxycholate.
The combination of lysostaphin and penicillin also exhibits synergy such that a 1000 fold increase in the killing of staphylococci is observed in vitro.
Infusions of a therapeutically effective amount of lysostaphin, with or without surfactant or EDTA, but with penicillin or said other antibiotic potentiating agents are used to achieve elimination of the staphylococcal infection. Whereas such infusions contain between 2 to 400mg lysostaphin when no potentiating agents are present, the required effective doses of lysostaphin can be lowered in the combinations used in this invention (as a result of their synergistically enhanced activity) by as much as 1000-fold.
Synergistic bactericidal activity of lysostaphin and penicillin was observed even upon administration to penicillinase-positive S. aureus and methicillin-resistant S. aureus ("MRSA"). MRSA are usually resistant to multiple antibiotics and are particularly problematic, especially in humans, as well as difficult to kill. The lysostaphin/penicillin combinations would be indicated for use in specific situations where grave MRSA infection cannot be controlled by conventional antibiotic (e.g. penicillin) therapy. In addition, penicillin or other similar acting substances (cell wall-active antibiotics) may also be useful together with lysostaphin as an agent against staphylococcal infection and contamination.
While the utility of the lysostaphin-containing formulations according to the invention is illustrated using mastitis treatment, the enhanced effectiveness of the lysostaphin in these formulations makes them suitable for a number of other applications involving staphylococcal infections and contamination. Thus, the formulations could be used to control staphylococcal infections by incorporating them into wound dressings and medications, disinfectant scrubs, wipes or lotions, or in surgical implants. The formulations might also he used for cleaning of medical instruments, and of floors, walls, bedding and the like in circumstances where environmental disinfection is desired. Other potential uses include use as a nasal infusion to reduce intra-nasal carriage of staphylococci, and food related uses such as treatment of meat, eggs, cheese and fish or food packaging and handling equipment.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1 shows a chromatogram of lysostaphin produced by transformant B. sphaericus strain 00 containing the recombinant plasmid pBC16-1L which codes for lysostaphin.

DETAILED DESCRIPTION OF THE INVENTION

Lysostaphin for use according to the claimed invention can be obtained from either natural or recombinant sources. Preferably, the lysostaphin is obtained from Bacillus sphaericus strain 00 containing a recombinant plasmid which directs the synthesis of lysostaphin, as this provides for both high levels of lysostaphin production substantially free from staphylococcal immunogenic contaminants and facile lysostaphin purification since the lysostaphin accumulates directly in the growth medium. Bacillus sphaericus transformants containing the plasmid pBC16-1L have been found to be particularly suited for this purpose, although other strains are also useful as a source of lysostaphin. One method for obtaining lysostaphin from microorganisms transformed by recombinant plasmids containing the gene which codes for lysostaphin is fully disclosed in U.S. patent application 034,464, filed April 10, 1987, which is a continuation-in-part of U.S. application 852,407. Both applications are incorporated herein by reference.

Method of Treatment

Prophylactic compositions for treating bovine mastitis according to the invention involve the use of lysostaphin/cell wall-active antibiotic-containing teat dips. Such lysostaphin-containing teat dips provide effective prevention of bovine mastitis when used before and after every milking. Preferably, the preventative regimen is used for all cows in the herd. Preferably, the teat dips comprise about 1.0 µg/ml lysostaphin in an acceptable carrier. In addition, teat dips for use according to the invention may include about 1.0 µg/ml mutanolysin, about 10 µg/ml lysozyme, and a mild surfactant. Acceptable carriers are those which provide a buffered medium of approximately pH 8.0 and include aqueous buffers or hydrophilic ointment bases. For example non-ionic detergents, fatty acids or other mild surfactants, protein carriers, such as serum albumin or gelatin, powdered cellulose and carmel can be used as a carrier. The teat dip according to the invention may also advantageously include chelating agents, such as EDTA, colorants, and humectants, such as glycerol or sorbitol.
Mutanolysin is obtained from Streptomyces globisporus. Lysozyme is obtained from chicken egg whites.
Intramammary infusion of lysostaphin can be used to effectively treat infected animals who have developed either chronic or acute staphylococcal bovine mastitis despite prophylactic treatment. A single dose of from 2 to 400 mg lysostaphin per milk gland will eliminate the infection and cure staphylococcal mastitis in most instances. Additional doses of lysostaphin may be indicated where the infection is persistent. Doses significantly higher than 400 mg are not recommended as they can lead to unwanted and potentially adverse side effects including transient swelling, tenderness, and reduced milk production. These effects are limited to the treated gland, however, so that higher doses to a single gland may be appropriate in severe and life-threatening situations. In life-threatening cases, the route of administration could also include sites other than the infected gland so as to achieve systemic delivery, i.e., intravenous, subcutaneous, or intramuscular, and rectal or oral administration of suitably encapsulated formulations in which the lysostaphin is protected from inactivation in the gut.
It has also been found that infusion of a combination of lysostaphin and penicillin is surprisingly much more efficacious than lysostaphin alone because of an apparent synergistically enhanced bactericidal activity of this combination. It is believed that the therapeutic compositions may include other cell wall-active antibiotic agents which potentiate the bactericidal activity of lysostaphin, for example, synthetic penicillins. The compositions can also contain chelating agents, mild surfactants, (e.g., deoxycholate) and other membrane active agents which may facilitate penetration of lysostaphin to the site of infection. In formulations that include e.g., penicillin, the dosage of lysostaphin can be decreased as a result of the potentiated bactericidal activity of lysostaphin. Since too high a dose of lysostaphin can induce unwanted and potentially adverse side-effects, this synergistic effect is significant not only for efficacy but also for avoidance of potential side effects. In the following illustrative examples, the words "Tween" and "Triton" are registered trade marks.

Examples 1-4

In vitro experiments were conducted to determine the bactericidal activity of lysostaphin, mutanolysin, and lysozyme compositions toward S. aureus and other mastitis pathogens. The protocol was as follows:

Protocol for Viable Cell Assays

1. Bacterial cells (generally 10⁹ cells/ml) from an overnight plate (incubated at 37°C) were resuspended in Tris buffer (20mM Tris, pH 8).
2. 10 µl of bacterial cell suspension and 1 ml of control and teat dip test formulation (i.e. milk, buffer, or buffered detergent etc., containing the lysostaphin composition) were combined.
3. The cells were incubated for various times at 37°C.
4. The bacterial suspensions were centrifuged for 2 minutes in benchtop microcentrifuge to pellet cells.
5. The pellet was washed twice with 1.0 ml Phage buffer.
6. The cells were resuspended in 1.0 ml of Phage buffer, serially diluted in Phage buffer as appropriate, and 100 µl were plated on GL agar (S. aureus, E. coli, Klebsiella pneumoniae.) or Trypticase Soy agar (S. agalactiae).
7. The plates were incubated overnight at 37°C and control and test plates were scored for colony forming units, (hereinafter CFU), to determine percent survival.

Composition of Phage buffer:

50 mM Tris, pH 7.8; 1 mM MgSO₄; 4 mM CaCl₂; 100 mM NaCl; Gelatin, 1.0 g/l. (Phage buffer helps stabilize any protoplasts and spheroplasts that did not lyse during treatment).

Composition of GL agar per liter:

Difco casamino acids, 3.0 g; Difco yeast extract, 3.0 g; NaCl, 5.9 g; Na lactate (60% w/v), 3.3 ml; 25% (v/v) glycerol, 4.0 ml; agar, 15 g; pH adjusted to 7.8.

Composition of Trypticase Soy agar per liter:

Bacto Tryptone, 15 g; Bacto Soytone, 5 g; NaCl, 5 g; agar, 15 g; pH adjusted to pH 7.3.
The results of in vitro experiments demonstrating the bactericidal efficacy of various lysostaphin therapeutic formulations are presented in Tables IA-IC. The results are presented as the percent survivals for S. aureus strains Newbould 305, strain RN451, the penicillin resistant strains RN1753 (Penicillinase producer) and Col strain (methicillin resistant).
Table IA presents results for formulations containing 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml and 0.00 µg/ml (CNTRL) lysostaphin. As can be seen from these results all levels of lysostaphin tested were effective to kill the organisms in a buffer vehicle (50 mM Tris, pH 8.0). In a milk vehicle, only 1 µg/ml and 0.1 µg/ml reduced bacterial survival.
Table IB shows the effect of adding a mild non-ionic surfactant, octylphenoxyl polyethoxy (10) ethanol, (Triton X-100), to the lysostaphin formulation. For example, less than 0.001% of the cells survive exposure to 0.1 µg/ml lysostaphin and 0.1% Triton X-100, while 2.2% and 7.7%, respectively, survived exposure to each compound alone. Even more surprising, less than 0.001% survival was observed for 0.01 µg/ml lysostaphin and 0.1% Triton X-100.
Table IC demonstrates the synergistic effect of lysostaphin/penicillin combinations on three strains of staphylococci. Depending on the doses of each, the combinations of lysostaphin plus penicillin can be 100 to 1000 times more effective than either lysostaphin or penicillin alone with all three strains.

Table ID demonstrates the effect of the combination of lysostaphin and penicillin compared with their sequential effect on S. aureus. S. aureus were suspended at 10⁷ cells/ml in milk and incubated for the times indicated in the table with either lysostaphin and penicillin together or sequentially. After incubation, samples were centrifuged to obtain cell pellets which were washed twice, resuspended in 1.0 ml Phage buffer, diluted and 100 µl plated on GL agar. Colony forming units (CFU) were scored after incubation overnight at 37°C to determine percent survival relative to appropriate controls. The lysostaphin/penicillin combination, exhibits a synergistically enhanced bactericidal activity against S. aureus which is at least 3 orders of magnitude greater than that seen when the two agents are added sequentially.

TABLE IA

The Effect of Lysostaphin On The Viability of S. Aureus
Strain	Vehicle	Incubation Time	% Survival
			1.0L	0.1L	0.01L	CNTRL
S. aureus Newbould 305	Milk	15'	2.8	75.0	100	100
S. aureus Newbould 305	Milk	2h	0.1	82.0	100	100
RN451	Milk	15'	<0.1	22	100	100
	Milk	2h	<0.01	41	100	100
	Buffer	2h	nd	2.2	20	100

TABLE IB

The Effect Of Non-Ionic Detergent On The Bactericidal Activity of Lysostaphin Toward S. aureus
Strain	Vehicle	Incubation Time	% Survival
			0.1L	0.01L	0.1%T	0.1L	0.01L	CNTRL
						+0.1%T+0.1%T
S. aureus RN451	Buffer +0.1% Triton	2h	2.2	20	7.7	<0.001	<0.001	100

TABLE ID

A Comparison of the Effect of the Combination of Lysostaphin and Penicillin Versus Their Sequential Effects on the Survival of Staphylcoccus aureus (Strain RN451) in milk at 37°C
	combo(2h)	lspn(2h)	pen(2h)	Pen(2h)/lspn(0.5h)	lspn(2h)/pen(0.5h)
%survival	0.0005	23	25	0.3	10
lspn = lysostaphin; pen = penicillin

In addition, assays for lysostaphin, mutanolysin, and lysozyme activities which measure the decrease in turbidity at 600 nm of suspensions of live S. aureus, S. agalactiae, and E. coli or Klebsiella pneumoniae, respectively, indicated that chelating agents (e.g., EDTA) potentiate the lytic activity of each of the three bacteriolytic enzymes.
The data indicate that lysostaphin is a rapidly acting, highly effective staphylocide, the bactericidal activity of which is potentiated more than 1000 times by penicillin or the mild surfactant, Triton® X-100. The inclusion of a chelating agent further potentiates the bactericidal activity of lysostaphin. It is also believed that synthetic penicillins and cell wall-active antibiotics will potentiate the activity of lysostaphin. Lysostaphin is an effective staphylocide in milk, but in buffer the bactericidal activity of lysostaphin is approximately 10 times that observed in milk.

Example 5

According to the general protocol described in Examples 1-4, further in vitro experiments were performed to evaluate the bactericidal activity of a lysostaphin composition comprising bacteriolytic enzymes, a non-ionic detergent and buffered chelating agent. As shown in Table II a formulation containing 1% Triton® X-100, 0.1 µg/ml lysostaphin, 10 µg/ml lysozyme, and 5 mM EDTA in 20 mM Tris, pH 8.0, (AMBI Teat Dip-0.1) was extremely effective against a wide range of mastitis-causing pathogens, including S. aureus strain Newbould 305, S. epidermidis, Streptococcus agalactiae strain McDonald and strain C48, and clinical isolates of Streptococcus uberis, E. coli, and Klebsiella pneumoniae.

TABLE II

In Vitro Efficacy Of AMBI Teat Dip-1 Against Mastitis Pathogens
Strain	Viable Count	% Survival
Staphylococcus aureus (Newbould 305)	7.0 x 10⁵	<0.001
Staphylococcus aureus (RN451)	5.7 x 10⁵	<0.001
Staphylococcus epidermidis (PS)	8.3 x 10⁵	<0.001
Streptococcus agalactiae (McDonald)	3.9 x 10⁵	<0.001
Streptococcus agalactiae (C48)	2.9 x 10⁴	<0.001
Streptococcus uberis (PS)	6.9 x 10⁵	<0.001
Escherichia coli (PS)	9.1 x 10⁵	<1.0
Klebsiella pneumoniae (PS)	9.6 x 10⁵	<1.0

Example 6

Trials on cows were performed which demonstrated the efficacy of lysostaphin teat-dip compositions in vivo. The tests were performed generally according to Protocol A of the National Mastitis Council. In general, teats were cleaned with a 1% iodine wash solution and dried with a paper towel. Teats were then rinsed with alcohol and allowed to air dry. All four teats per cow were next dipped in a 10⁸ cell/ml suspension of S. aureus strain Newbould 305 to cover 1/2 the teat, and allowed to air dry for 30 minutes. Two teats (right fore and left rear) were then dipped in a lysostaphin test teat dip formulation (10 µg/ml lysostaphin in 0.85% saline) to cover 2/3 of the teat, and allowed to air dry for 30 minutes; the remaining two teats acted as non-treated controls. Each teat was first swabbed with a moist cotton swab and then washed with 10 ml of 0.85% sterile saline solution; the wash was collected into a sterile 30 ml tube. A 0.2 ml sample of the wash, and appropriate dilutions thereof, were plated on blood agar in duplicate and incubated at 37°C for 24-48 hours, Colony forming units were determined and percent survival of S. aureus calculated relative to controls.
Ten µg/ml solutions of lysostaphin in 0.85% saline completely disinfected invading S. aureus from cow teat surfaces. Moreover, lysostaphin applied to teat surfaces prior to exposure of teats to S. aureus suspensions had sufficient residual activity on the teat surface to prevent colonization of the teat. Residual activity could be enhanced by inclusion of a polymeric adsorbent and/or inert carried protein to reduce lysostaphin wash-off.

Example 7

In accordance with the results from Example 6 and the data obtained in vitro, an enhanced teat dip formulation (AMBI Teat Dip 1.0) comprising 1.0 µg/ml lysostaphin, 10 µg/ml lysozyme, 1.0 % Triton® X-100, and 5 mM EDTA in 20 mM Tris buffer, pH 8.0 was evaluated as a disinfectant against S. aureus strain Newbould 305. Teats were dipped in 10⁸ cells/ml S. aureus strain Newbould 305, and allowed to air dry for 30 min. The treated teats were then dipped in AMBI test teat dip-1.0 solution (1.0 ug/ml lysostaphin, 10.0 µg/ml lysozyme, 1.0% Triton® X-100, 5 mM EDTA, 20 mM Tris buffer, pH 8.0) and allowed to air dry for 30 min. Teats were swabbed with a moist cotton swab, and rinsed with 10 ml sterile 0.85% saline. The swab and rinse were plated separately on blood agar plates, incubated 24-48h and CFU determined. The results, shown in Table IIIA clearly demonstrate the efficacy of this preparation. At least a 3 fold order of magnitude reduction was observed in the numbers of S. aureus recovered from treated teats; 50% of treated teats were free from invading S. aureus.

Corresponding tests were performed in which teats were dipped in preparations containing 2 x 10⁷ cells/ml Streptococcus agalactiae strain McDonald, and then allowed to air dry for thirty minutes. The results of these tests are shown in Table IIIB. All of the treated teats were free of S. agalactiae.

TABLE IIIA

In Vivo Efficacy of AMBI Teat Dip-1.0 Against Staphylococcus aureus On Cow Teats
Cow No.	CONTROL CFU's per ml		CONTROL CFU's per ml
	LF	RH	LH	RF
1	225	1,675	13	0
2	24,500	19,500	8	175
3	300	15,000	0	15
4	78	155	0	150
5	50,500	18,750	5	8
6	44,250	65,500	0	0
7	75	43	35	3
8	175	1,150	0	0
9	68	58	0	5
10	550	300	0	0
Average	12,072	12,213	6	36
Total Qtrs Negative	0/10	0/10	6/10	4/10

TABLE IIIB

In Vivo Efficacy of Teat Dip-1.0 against Streptococcus agalactiae (McDonald strain) on Cow Teats
Cow. No.	CONTROL CFU's per ml		TREATED CFU's per ml
	LF	RH	LH	RF
1.	5	15	0	0
2.	53	360	0	0
3.	115	48	0	0
4.	150	10	0	0
5.	13,750	1,200	0	0
6.	16,250	725	0	0
7.	95	320	0	0
8.	0	450	0	0
9.	1,175	775	0	0
10.	150	300	0	0
Average	2,574	420	0	0
Total Qtrs negative:	1/10	0/10	10/10	10/10

Example 8

Guinea pig mammary glands were infected with 200-300 CFU of S. aureus strain Newbould 305. Three days post-infection, the glands were infused with a single dose of lysostaphin dissolved in 200 µl 0.85% sterile saline. Milk samples were collected from the glands 6 hours after treatment and at 12 hour intervals thereafter for at least 5 days after treatment. 100 µl milk samples from treated and nontreated infected glands were plated on blood agar. After 24-48 hours incubation, the plates were counted to determine CFU. The single doses of lysostaphin which were sufficient to eliminate the infection did not produce adverse side effects and indicated that intramammary infusions of lysostaphin are effective against staphylococcal mastitis. At 125 µg/kg, glands were cleared of infection by the 6 hour post-treatment sample and remained clear throughout the study.

TABLE IV

Efficacy of Intramammary Infusion of Lysostaphin Toward Experimental STAPHYLOCOCCAL Mastitis in Guinea Pig
	Lysostaphin Dose µg/kg
	ZERO	1.0	5.0	25.0	62.5	125.0
Number of animals cleared of infection	(0/10)	(1/0)	(1/2)	(2/2)	(1/1)	(7/7)

It can be seen from these examples that lysostaphin is effective for treatment of staphylococcal mastitis and that its effect is greatly enhanced when used in combination with penicillin or with substances such as mild surfactants and chelating agents.

Production of Lysostaphin from Bacillus

Lysostaphin for use according to the claimed invention can be obtained from either natural or recombinant sources. Preferably, the lysostaphin is obtained from cultures derived from Bacillus sphaericus strain 00 transformed by recombinant plasmids which direct lysostaphin synthesis as described in copending application Serial No. 034,464 filed April 10, 1987 which is a continuation-in-part of Serial No. 852,407 filed April 16, 1986. This method provides for both high levels of lysostaphin production substantially free from staphyloccal immunogenic contaminants. Lysostaphin purification is facilitated since active lysostaphin accumulates directly in the growth medium. Using this method, Bacillus sphaericus 00 transformants containing plasmid pBC16-1L (B. sphaericus 00/pBC16-1L) have been found to be particularly suited for the purpose, although other transformed Bacillus strains are also useful as a source of lysostaphin.

The lysostaphin-producing organism is grown under conditions conducive to the production of lysostaphin. The optimum conditions will vary from strain to strain; however, certain types of growth media and fermentation conditions are known to enhance lysostaphin production. In the case of the Bacillus sphaericus 00/pBC16-1L transformant, the preferred growth medium is VY broth (25g Veal Infusion + 5g Yeast Extract/liter) under well-aerated conditions (see Table V).

TABLE V

Effect of Aeration of Lysostaphin Production by the Bacillus Sphaericus 00/pBC 16-1L Transformant
Klett	Stirring Speed
	100 rpm	200 rpm	200 rpm (Fluted)	320 rpm
250	21.8	36.2	35.9	30.0
350	40.1	68.9	45.3	45.0
400	88.5	62.7	102.8	71.4
450	n/a	86.4	52.3	135.9
O/N	64.4	31.3	37.6	57.5

Cultures (40 ml) in 300 ml Klett flasks were inoculated with 4 ml of overnight primary culture. Growth medium: VY broth containing 5 µg/ml erythromycin.
Samples were removed at times throughout growth. Supernatants were assayed for lysostaphin activity by turbi-dometric clearing of dead cell suspensions of S. aureus. Results are presented as µg lysostaphin per ml. B. sphaericus 00/pBC16-1L transformant grown on VY medium produced and secreted approximately 130 mg lysostaphin per liter of culture medium, which is more than four times the amount produced by S. simulans under the best fermentation conditions currently available. Lysostaphin accumulates in the growth medium with little or no degradation, even after prolonged incubation of cultures, and accounts for more than 80% of total extracellular protein.
Lysostaphin is isolated from the growth medium in accordance with known fractional precipitation (salting out) procedures. Alternatively, a particularly effective purification is achieved by combining a precipitation and a chromatographic separation of the fermentation broth from cultures of the lysostaphin-producing B. sphaericus 00/pBC16-1L transformant.
Cells are removed from the fermentation broth, for example by centrifugation or ultrafiltration, and solid ammonium sulfate is added to the supernatant to 40-60%, preferably 50%, of saturation. After 1 hour at 4°C, the lysostaphin-containing precipitate is recovered by centrifugation. Recovery at this step is greater than 80%.
The precipitate is redissolved in a minimum volume of 10 mM sodium phosphate buffer (pH 7.00, 50 mM NaCl) and dialyzed against 100 volumes of the same buffer. After removal of any particulate material, the dialyzed solution is chromatographed on a cation exchange column (preferably Pharmacia FPLC Mono S) and eluted using a buffered gradient of increasing salt concentration from 0.05 to 0.25 M NaCl. Recovery of lysostaphin for the single chromatographic step was more than 90%. Lysostaphin activity is associated with two major peaks (Fig. 1). The later eluting peak of lysostaphin is comprised of non-covalent aggregates of the protein. These aggregates dissociate on dilution in buffer and under conditions of sodium dodecylsulfate polyacrylamide gel electrophoresis.

Construction of the plasmid vector pBC16-1L which contains the gene coding for lysostaphin

Lysostaphin-producing strains of Bacillus sphaericus can be produced using recombinant DNA techniques and preferably those described in copending applications 852,407 and 034,464. Specifically, total S. simulans DNA is partially cleaved by the appropriate restriction endonuclease and DNA fragments so generated are then ligated to a linearized known vector (pUC8) with compatible ends, carrying an antibiotic resistance marker and the lac Z′ gene (i.e. β-galactosidase gene). The ligation mix is then transferred to E. coli (JM105) by transformation. Successful insertions of the lysostaphin gene into the plasmid can be found by selecting for transformants by growth on the appropriate antibiotic, and then finding those with a lac Z′ negative phenotype. Lysostaphin production is detected by turbidometric clearing of a suspension of S. aureus either in solution format or as an overlay on agar plates.
Using various lysostaphin-producing E. coli JM105 transformants, restriction analysis and subcloning of the JM105 plasmid DNA showed that the DNA sequence coding for lysostaphin was localized to a 1.5 kbp Hpa II-Hind III DNA fragment. This fragment was visualized after electrophoresis by ethidium bromide staining and transferred to a nitrocellulose filter strip. The strip was washed with NET buffer (0.15 M NaCl, 0.1 m EDTA, 0.02 M Tris, pH 8.0) and the transferred DNA was eluted by incubation of the strip in NET buffer containing 1 M NaCl for 1 hour at 65°C, Ethidium bromide was removed from the DNA by extraction with n-butanol. DNA, precipitated by addition of two volumes of cold 95% ethanol to the aqueous phase, was collected by centrifugation, washed with 80% ethanol and dissolved in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). Recombinant plasmids capable of transforming B. subtilis as well as B. sphaericus to express lysostaphin were constructed using a derivative of plasmid pBC16 (pBC16-1) as a cloning vector. pBC16 is a tetracycline resistant (Tet^r) Bacillus plasmid, originally isolated from B. cereus (K. Bernhard, H. Schremph, and W. Goebel, 133 J. Bact. 897, 1978). Plasmids indistinguishable from pBC16 by restriction analysis and Southern Hybridization were also found in soil isolates of B. subtilis and B. sphaericus (J. Polak and R.N. Novick, 7 Plasmid 152, 1982).
The pBC16 derivative (pBC16-1) used as the cloning vector was constructed by ligating the TaqIA fragment of plasmid pE194 (B. Weisblum, M.Y. Graham, T. Gryczan, and D. Dubnau, 137 J. Bact. 635, 1979), an erythromycin resistant (erm^r) plasmid from S. aureus, with a partial TaqI digest of plasmid pBC16 using T4 Ligase. After transfer of the ligation mixture to B. subtilis by protoplast transformation (S. Chang and S.N. Cohen, 168 Molec. Gen. Genet. 111, 1979), clones that were resistant to both tetracycline and erythromycin were selected. One such clone was designated pBC16-1.
Restriction analysis revealed that pBC16-1 contained all of the pBC16 TaqI fragments plus the TaqIA fragment of pE194 which contains the erythromycin resistance determinant. pBC16-1 was then digested with the restriction endonuclease PvuII, thereby removing about 25% of the plasmid DNA including most of the tetracycline resistance determinant. The Pvu II-digested vector pBC16-1 was treated with calf intestinal alkaline phosphatase. The 1.5 Kbp DNA fragment which codes for lysostaphin was treated with the Klenow fragment of DNA polymerase. The 1.5 Kbp DNA fragment and plasmid DNA were then mixed and ligated using T4 ligase, and the ligation mixture was transferred to B. subtilis by protoplast transformation. Transformants were resistant to erythromycin, sensitive to tetracycline, and produced lysostaphin as indicated by zones of clearing when grown on agar containing dead S. aureus cells. One such lysostaphin producing clone was picked and designated B. subtilis/pBC16-1L. Plasmid pBC16-1L DNA extracted from the B. subtilis/pBC16-1L transformant was isolated after ultracentrifugation in an ethidium-bromide cesium chloride density gradient. Plasmid pBC16-1L DNA was transferred by protoplast transformation to various species of Bacillus, including B. sphaericus strain 00. Transformants were resistant to erythromycin and produced lysostaphin. The B. sphaericus 00/pBC16-1L transformant provides maximum production of lysostaphin and permit accumulation of intact, enzymically active product. B. sphaericus strain 00 was originally isolated from soil and is maintained in the culture collection (RN3106) of the Public Health Research Institute, New York, New York. B. sphaericus 00/pBC16-1L is maintained in the culture collection of the Public Health Research Institute, New York, New York and has been deposited with the American Type Culture Collection under ATCC Accession No. 67398.

Claims

A lysostaphin-containing composition for killing staphylococci (and which therefore does not itself contain staphylococci), characterized in that it also comprises at least one cell wall-active antibiotic in an amount effective synergistically to enhance the bactericidal effect of lysostaphin against staphylococcal mastitis.
A composition according to Claim 1, characterized in that the antibiotic is penicillin.
A composition according to Claim 2, characterized in that the penicillin is present in a concentration of 0.1 to 10.0 micrograms/ml.
A composition according to Claim 1, characterized in that the antibiotic is a synthetic penicillin.
A composition according to any preceding Claim, characterized in that the lysostaphin is present in a concentration of at least 0.01 microgram/ml.
A composition according to any preceding Claim, characterized in that it further comprises a mild surfactant.
A composition according to Claim 6, characterized in that the surfactant is present in a concentration of from 0.1 to 1.0 percent.
A composition according to any preceding Claim, characterized in that it further comprises a chelating agent.
A composition according to any preceding Claim, characterized in that it further comprises at least one additional bacteriolytic agent.
A composition according to any preceding Claim, characterized in that it further comprises mutanolysin and lysozyme.
A composition according to any preceding Claim, characterized in that the lysostaphin is derived from a transformant microorganism containing a recombinant plasmid which codes for lysostaphin.
A composition according to Claim 11, characterized in that the recombinant plasmid is that designated pBC16-1L and present in cells of Bacillus sphaericus strain 00 ATCC 67398.
A composition according to any preceding Claim, characterized in that it further comprises an acceptable carrier.
A composition according to any preceding Claim, for use in the treatment or prevention of staphylococcal mastitis.
A composition according to any one of Claims 1 to 13, for use as a teat dip in the prevention of bovine staphylococcal mastitis.